This invention relates to new purine nucleoside 5'-phosphate (mono, di or tri) 3' (2')-diphosphates and the lithium, sodium and potassium salts thereof, and to methods for the production of the purine nucleoside 5'-phosphate (mono, di or tri) 3' (2')-diphosphates. More particularly, it relates to new purine nucleoside 5'-phosphate (mono, di or tri) 3' (2')-diphosphates which are produced by the enzymatic transfer of pyrophosphoryl group from ATP, dATP and pppApp to specific 5'-purine nucleotides using microorganisms belonging to the genera Streptomyces, Actinomyces and Streptoverticillium, or a new nucleotide pyrophosphotransferase thereof, and to processes for the production of the purine nucleoside 5'-phosphate (mono, di or tri) 3' (2')-diphosphates. It also relates to a new nucleotide pyrophosphotransferase, and to the methods for its recovery and purification from the cultured broth and mycelium of microorganisms belonging to the genera Streptomyces, Streptoverticillium and Actinomyces.
As is well known, adenosine tetraphosphate (ppppA), guanosine tetraphosphate (ppGpp) and Guanosine pentaphosphate (pppGpp) are distributed in nature, especially in microorganisms and animals, as unusual nucleoside phosphates. The ppGpp and pppGpp are particularly being watched with keen interest in the regulation of protein and ribonucleic acid syntheses in E. coli, and it is expected that they have an important role in glycolysis, lipid synthesis, energy metabolism and phosphorylation of various organisms.
In the present invention, purine nucleoside 5'-phosphate (mono, di or tri) 3' (2')-diphosphate compounds are generalized as the following symbol: EQU mXpp
in which X represents one of the nucleosides selected from adenosine (abbreviated as A), guanosine (abbreviated as G) and inosine (abbreviated as I), and p and p (CH.sub.2)p stands for phosphate residue and methylene phosphate residue, respectively. In this specification phosphate residue is also referred to as phosphoryl group.
The notation, m represents a number of phosphoryl groups at the 5'-position of the nucleoside in which m is an integral number from 1 to 3. The p after the X is the 3' (2')-position. The notation 3' (2')-indicates that the position of the phosphoryl group attached to the ribose is either 3'- or 2'- of the ribose, as there is the reversible exchange of the phosphoryl group between the 3'- and the 2'- position of ribose in the nucleoside under a certain condition. For instance, pppApp indicates adenosine 5'-triphosphate 3' (2')-diphosphate and p(CH.sub.2)ppGp is .crclbar.,.gamma.-methylene guanosine 5'-triphosphate 3' (2')-diphosphate.
However, it is difficult to extract unusual purine nucleoside phosphates such as ppGpp and pppGpp from microorganisms because of a very small distribution therein, and further the chemical synthesis of these nucleoside phosphates is not established yet. Biochemical syntheses of ppGpp and pppGpp are possible using E. coli ribosome, ATP and GDP or GTP, but this method is not suitable for industrial production of large amounts, because of the complicated processes and extremely low yield. Other new nucleoside 5'-phosphate (mono, di or tri) 3' (2')- diphosphates such as pppApp, ppApp, pppIpp, etc. cannot be produced by E. coli. In order to investigate profoundly the physiological role of these nucleotides in organisms, development of an economical and simple method for their preparation has been awaited. ATP is the most important substance in phosphorylation and energy metabolism of organisms, and recently it was shown that E. coli and B. subtilis can synthesize enzymatically the characteristic nucleotides, ppGpp and pppGpp, which participate in nucleic acid and protein syntheses, from ATP and GDP or GTP. However, the purification of the enzyme playing a part in the formation of ppGpp and pppGpp has been unsuccessful so far. A number of phosphotransferases and nucleotide-degrading enzymes have been isolated from microorganisms and characterized, but the enzyme concerned with the formation of the aforementioned nucleoside phosphates has not been reported yet.